Architecture and Dynamics of Biological Macromolecules, Institut Pasteur, Université Paris Cité, CNRS, UMR 3528, Paris, France.
Univ Brest, Ifremer, CNRS, Biologie et Ecologie des Ecoystèmes marins profonds (BEEP), F-29280, Plouzané, France.
Nat Commun. 2023 Apr 22;14(1):2326. doi: 10.1038/s41467-023-38048-w.
Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.
复制蛋白 A（RPA）是一种异源三聚体单链 DNA 结合蛋白，在 DNA 复制、重组和修复中具有重要作用。关于生命第三域古菌中的 RPA 结构知之甚少。通过采用综合的结构、生化和生物物理方法，我们在存在和不存在 DNA 的情况下对 Pyrococcus abyssi 的 RPA 进行了广泛的表征。获得的 X 射线和 cryo-EM 结构表明，真核生物和古菌之间共享三聚体核心和促进 RPA 在 ssDNA 上聚集的相互作用。然而，我们还鉴定出一个名为 AROD（酸性 Rpa1 OB 结合结构域）的螺旋结构域，并表明在古菌中，RPA 在没有 DNA 的情况下形成出乎意料的四聚体超复合物。在四聚体中聚集的四个 RPA 分子可以有效地包裹和保护由前进的复制体产生的 ssDNA 片段。最后，我们的结果为真核生物中这种原始复制因子的进化提供了新的见解。
